P. vivax malaria.Angiogenic factors such as angiopoietin 1 (Ang-1) and angiopoietin 2 (Ang-2) are biomarkers produced during activation and dysfunction of the vascular endothelium in several infectious diseases. The aim of this study was to determine the serum levels of Ang-1 and Ang-2 and to establish their relationship with the main indicators of worst-case prognosis in patients with This is a retrospective case-control study nested within a cohort of symptomatic malaria patients. A potentially severe case was defined as a patient that presented at least one of the main indicators of the worst-case prognosis for falciparum malaria, as established by the World Health Organization. Ang-2 and Ang-1 and the Ang-2/Ang-1 ratio were used to analyze the role of angiopoietins as biomarkers in signaling potentially severe vivax malaria. ROC curves were generated to identify a cut-off point discriminating between the angiopoietin concentrations that were most strongly associated with potential infection severity.P. vivax malarial infection, was larger in the subgroup of patients with platelet counts <75,000/µL.The serum levels of Ang-2 and the Ang-2/Ang-1 ratio were higher in the case group. In contrast, the serum levels of Ang-1 were lower in the cases than in the control patients. The blood count for platelets showed a positive correlation with Ang-1 and a negative correlation with Ang-2 and with the Ang-2/Ang-1 ratio. The area under the ROC curve (AUC) for serum angiopoietins, as an indicator of worst-case prognosis in a potentially severe P. vivax malaria have lower Ang-1 and higher Ang-2 serum levels  than controls. Elevated serum levels of Ang-2 and high values for the Ang-2/Ang-1 ratio may potentially be used as predictors of worst-case prognoses for P. vivax malaria, especially in patients with thrombocytopenia.This study showed that patients with predictors of worst-case prognoses for  Plasmodium vivax malaria has increased, moving away from the conventional idea that this infection is benign P. vivax malaria, multiple syndromes have been described, similar to those caused by P. falciparumP. vivax as those proposed by the World Health Organization P. falciparumOver the past few years, the number of publications on severe P. falciparum, P. vivax has a greater capacity to induce an inflammatory response, which results in a lower pyrogenic threshold P. falciparum, are less frequent in severe vivax malaria P. falciparum and severe infections caused by P. vivax, of which severe anemia Compared to P. vivax have been described, including those for endothelial lesions, hemolysis, and oxidative stress. Numerous substances have been evaluated as biomarkers for the severity of malarial infections such as proteins associated with iron metabolism and anti-oxidant enzymes, as well as various cytokines and chemokines P. vivax infections, and its association with severe cases has been established A number of laboratory markers have been used to assess inflammatory patterns in response to malaria and their relation to the severity of the infection An understanding of the relationship between the presentation of the disease and the production of a biomarker may help in clinical decision-making and could minimize the risk of disease complications and consequently, the suffering of patients P. falciparum infections, as well as that between the levels of these proteins and the occurrence of cerebral malaria, placental malaria, retinopathy, and death P. vivax infections P. vivax malaria.Recent studies have elucidated the relationship between disease severity and plasma levels of angiopoietins in This retrospective case-control study was performed within a cohort of malaria patients treated at the Hospital Julio Müller, in the city of Cuiabá, state of Mato Grosso, Brazil, between December 2011 and November 2013. The hospital is a reference center for the diagnosis and treatment of malaria in people living and traveling in the south and southeast of the Brazilian Amazon. Participation in the study was voluntary, with a free and informed consent form signed by the patient, or in the case of children, by the parents. This study was approved by the Ethics Committee of the Julio Müller School Hospital (Document 130/CEP/HUJM/2011).P. vivax, as confirmed by microscopy of a thin blood smear and polymerase chain reaction (PCR) analysis. Following the confirmation of the diagnosis, the patients underwent clinical examination, as well as hematology and blood biochemistry tests, including those for serum levels of Ang-1 and Ang-2. To analyze the role of angiopoietin as a biomarker for potentially severe vivax malarial infections, a subgroup of patients were classified as potentially severe cases, if they presented at least one of the main indicators (for children and adults) of the worst-case prognosis for falciparum malaria, as established by the World Health Organization Patients with symptomatic malaria were eligible to participate in the study if they had a confirmed monoinfection with g. The serum was removed and aliquots were stored at −80°C. The serum was defrosted only once prior to the assay for angiopoietin.Ang-1 is present in platelet granules and is released upon platelet activation The hematological evaluation of the patients was performed using a Cell Dyn Ruby multi-parameter automated hematology analyzer . Blood biochemistry was analyzed by photometry using a BT-3000 Plus automated chemistry analyzer .Angiopoietin levels in the serum samples were assessed by enzyme-linked immunosorbent assay (ELISA) using standard commercial kits  for Ang-1, the Quantikine Angiopoietin-1 kit (Lot 306702), and the Quantikine ELISA Human Angiopoietin-2 kit (Lot 308323) for Ang-2, according to the manufacturer’s instructions.U test was used to compare the distribution of the classic indicators for potentially severe malarial infection between the cases and the control patients. Spearman’s correlation test was used to analyze the association between angiopoietins and platelet counts. Because of heteroscedasticity, we used the non-parametric Kruskal–Wallis test to compare the values of the levels of angiopoietin and classified the patients into three groups according to their platelet counts: severe thrombocytopenia , moderate thrombocytopenia , and no thrombocytopenia . Cuzick’s test for trend was used to analyze the tendency of quantitative variables between platelet counts of the different groups analyzed. We generated receiver operating characteristic (ROC) curves and calculated the area under the ROC curve (AUC) to evaluate the diagnostic accuracy of angiopoietins for discriminating between patients with and without the indicators of the worst-case prognosis for potentially severe vivax malaria. The Youden ‘J’ index was used to define the cut-off points corresponding to the sensitivity and specificity values with the lowest probability of having occurred randomly Because of the non-normality of the data, non-parametric statistics were used for the analysis. The Mann–Whitney P. vivax monoinfection were included in the study. Of the 80 patients, 58 (72.5%) were men and 22 (27.5%) were women, with ages ranging from 5 to 78 years, with a median (1st–3rd quartile) of 39 (26–49) years. All had presented with fever within the 24 h prior to entering the study. Altered hematologic and biochemical parameters were infrequent except for platelet counts, for which the median (1st–3rd quartile) was 98,000  platelets/µL for all patients. Severe and moderate thrombocytopenia were observed in 10 (12.5%) and 45 (56.2%) patients, respectively. Only 25 (31.3%) of the patients showed normal platelet counts. The clinical and laboratory characteristics of patients according to platelet count are shown in Eighty patients with symptomatic malaria caused by PCR-confirmed Eighteen patients were classified as potentially severe cases in this study, showing one or more of the main indicators of the worst-case prognosis for malaria: severe anemia (n = 1), elevated blood urea nitrogen (BUN) (n = 3), hyperbilirubinemia (n = 10), and elevated serum transaminases (n = 4). The ages of the potentially severe cases did not differ from those of the controls (P = 0.870). As expected, however, the parameters signaling severe malarial infection were altered in the potentially severe cases, with significant differences in serum bilirubin levels (P = 0.008), aspartate aminotransferase (AST) (P<0.001), alanine aminotransferase (ALT) (P = 0.002), and parasitemia (0.010). No significant differences in hemoglobin (P = 0.760), hematocrit (0.471), serum urea (0.179), serum creatinine (P = 0.061), platelet count (P = 0.097), or axillary temperature (P = 0.126) were observed between cases and controls .Angiopoietin levels were not associated with other factors known to be related to the severity of malaria, such as age, level of parasitemia, or axillary temperature. When the analysis of the serum levels of angiopoietin was stratified according to the patients’ platelet counts, we found that the median value of Ang-1 was lower (P = 0.0001) in the patients with thrombocytopenia than in those with normal platelet counts. The median values for Ang-2 and the Ang-2/Ang-1 ratio, on the other hand, were higher  in the patients with thrombocytopenia. As the platelet count increased, so did the levels of Ang-1 (P<0.001) but the levels of Ang-2 decreased (P = 0.003) as did the Ang-2/Ang-1 ratio (P<0.001). In summary, regardless of whether or not the patient met the worst-case prognosis criteria, the blood count for platelets showed a positive correlation with Ang-1  and a negative correlation with Ang-2  and with the Ang-2/Ang-1 ratio  .ROC curves were generated to identify a cut-off point to discriminate between angiopoietin concentrations that were most strongly associated with the potential severity of the case, including the subgroups of patients stratified according to the platelet blood count. From the ROC curve, a highly accurate cut-off level for serum angiopoietins was defined as an indicator for a worst-case prognosis of a potentially severe malarial infection. In the analysis of all patients, the AUC was 0.667 and the cut-off point for Ang-2 was 5.9 ng/mL (77.8% sensitivity and 50.0% specificity), whereas for the Ang-2/Ang-1 ratio, the AUC was 0.737 and the cut-off point was 0.75 (61.1% sensitivity and 83.9% specificity). Considering only the subgroup of patients with platelet counts <75,000/µL, the ROC curve showed an AUC of 0.833 and a cut-off point of 8.4 ng/mL for Ang-2 (87.5% sensitivity and 66.7% specificity), whereas for Ang-2/Ang-1, the AUC was 0.881, with a cut-off point of 1.2 (75.0% sensitivity and 90.5% specificity). In patients with platelet counts >75.000/µL, the AUC was 0.527 and the cut-off point was 3.85 ng/mL for Ang-2 (80.0% sensitivity and 29.3% specificity), whereas for Ang-2/Ang-1, the AUC was 0.645 and the cut-off point was 0.30 (60.0% sensitivity and 68.3% specificity) .P. vivax infections. In P. falciparum malaria, the production of angiogenic factors is principally associated with an increase in the cytoadherence of infected erythrocytes to the vascular endothelium P. vivax promotes cytoadhesion P. vivax infections remain unclear. Only two studies have described the relationship between angiopoietins and malaria caused by P. vivax, and these presented conflicting results. One report showed lower levels of Ang-2 in patients infected by P. vivax than in patients infected by P. falciparumP. vivaxIn this study, low levels of Ang-1 and high values of Ang-2 and the Ang-2/Ang-1 ratio were associated with criteria that indicate the worst-case prognosis in patients with symptomatic In vitro and in vivo studies have demonstrated that higher concentrations of Ang-2 are associated with elevated production of TNF-α, nitric oxide, vascular endothelial growth factor (VEGF), and hypoxia Angiopoietins are proteins that are intimately involved in the stability of the endothelium P. vivax malaria patients, whereas a negative correlation was detected between platelet counts and Ang-2 and Ang-2/Ang-1 levels. Although not a defining feature of severe malaria, vivax-associated thrombocytopenia occurs in more than 24% of patients with vivax malaria In this study, a positive correlation was observed between platelet counts and Ang-1 levels in P. vivax and the elevation of Ang-2 levels.The association between Ang-1, Ang-2, and the Ang-2/Ang-1 ratio was stronger in patients with thrombocytopenia. Some possible explanations for this association are given below. Firstly, Ang-1 is secreted by platelet granules, and thus its concentration in the plasma is dependent on the number of circulating platelets P. vivax malaria patients have not been identified to date. Disequilibrium in the expression and production of angiogenic factors is associated with the severity of malaria caused by P. falciparumP. falciparum malaria than lactate Although useful for clinical decision-making, biomarkers that predict the appearance of criteria indicating the worst-case prognosis for P. vivax malaria, especially in patients with low platelet counts. Once incorporated into routine hospital laboratory investigation, the systematic assay of angiopoietins could help isolate patients with the potential for developing severe malaria during clinical management of vivax malaria.To the best of our knowledge, this study provides the first evidence of the relationship between changes in angiopoietin levels and criteria indicating the worst-case prognosis for The main limitation of this study is the small number of patients examined, thus restricting the possibilities for extrapolating the results to other populations. It is also important to point out that the comorbidities of the patients studied may explain the findings suggesting a worst-case prognosis, as already shown in the literature P. vivax malarial infections have lower serum Ang-1 and higher serum Ang-2 levels  than controls. Furthermore, a positive correlation was observed between platelet count and Ang-1, and a negative correlation was observed between platelet count and Ang-2 (and the Ang-2/Ang-1 ratio). Elevated serum levels of Ang-2 and high values for the Ang-2/Ang-1 ratio may potentially be used as predictors of worst-case prognoses for P. vivax malaria, especially in patients with thrombocytopenia.This study showed that patients with worst-case prognoses for


The 11S storage globulin of white lupin seeds binds to a metal affinity chromatography matrix. Two unusual stretches of contiguous histidine residues, reminiscent of the multiple histidines forming metal binding motifs, at the C-terminal end of 11S globulin acidic chains were hypothesized as candidate elements responsible for the binding capacity. To prove this, the protein was incubated with a lupin seed endopeptidase previously shown to cleave at twin arginine motifs, recurrent in the sequence region of interest. Upon incubation with this enzyme, the loss of metal binding capacity paralleled that of the anti-his-tag reactive polypeptides. The recovered small proteolytic fragment was analyzed by mass spectrometry and N-terminal sequencing and found to correspond to the 24-mer region cleaved off at twin arginine residues and containing the natural his-tag-like region. Similarly, when lupin seeds were germinated for a few days, the his-tag containing 11S globulin chain was converted to a form devoid of such region, suggesting that this mechanism is a part of the natural degradatory process of the protein. The hypothesis that the ordered and controlled dismantling of storage proteins may generate peptide fragments with potential functional roles in plant ontogenesis is presented and discussed. Among them, the frequent presence of twin and multiple arginine residues in the amino acid sequence of several storage proteins has been proposed as a cleavage-prone site for selective degradation . A whiteentified . Althougentified , no atteentified  and it ientified , and theIn addition to such sequence peculiarity, various legume seed storage proteins, including soybean vicilin-like globulin , have beAnother scarcely investigated aspect of structure/function relationships in the seed storage proteins is the description at molecular level of the detailed proteolytic modifications and the orderly dismantling of these proteins during seed germination , 6 and tThis work aims at filling some of these gaps and contributing to a better knowledge of these processes by using a white lupin seed legumin-like globulin as a model storage protein source.Lupinus albus, L., var. Multitalia were a kind gift from Agroservice Spa . Anti-his-tag antibodies were purchased from GenScript ; antibodies to lupin 11S basic chain were prepared as described in ref. [Seeds of  in ref. . All othLupin 11S globulin was purified as described previously . BrieflyThe 11S globulin solution was loaded onto a preparative nickel affinity column (120 x 30 mm)  equilibrated with 50 mM Tris-HCl, pH 7.5, containing 0.5 M NaCl. The bound fraction was eluted with 0.25 M imidazole in the same buffer. For analytical purposes, 300 μg of 11S globulin sample were loaded onto a 5 mL NiNTA-HPLC column  equilibrated in 50 mM Tris-HCl pH 7.5 containing 0.5 M NaCl, at a flow rate of 1 mL/min and monitoring at 280 nm. After 12 minutes, the elution of his-rich peptides was carried out by a gradient of 0–25% (v/v) of 1 M imidazole for 2 minutes and maintaining 0.25 M imidazole concentration for further 10 minutes.RP-HPLC of the fraction eluted by NiNTA using imidazole was carried out on a Symmetry300 C18 column (4.6 × 250 mm) , equilibrated with 0.1% (w/v) trifluoroacetic acid in water at pH 2.2. The elution of the peptide fractions, at a flow rate of 0.8 mL/min, was carried out by a continuous gradient of 0–75% (v/v) acetonitrile for 75 min and monitored at 220 nm.3.Lupin seed -R-R- endopeptidase was prepared using a combination of size exclusion, ion-exchange, affinity and metal affinity chromatography according to ref. . For theg for 15 min and the proteins analyzed on SDS-PAGE as described in the next paragraph.Lupin seeds were vernalized at 4°C overnight and then germinated at 20°C for 24, 48, 72 and 96 hours. At the end of the incubation time, the seeds were dehulled, ground and the proteins extracted with SDS-PAGE denaturing solution containing 0.4% SDS and 2-mercaptoethanol in the ratio 1:20 w/v and immediately denatured at 100°C for 10 minutes. Extracts were centrifuged at 10,000 x SDS-PAGE was carried out according to Laemmli  on 12% p2O2 with 4-chloronaphthol as substrate.For immunodetection, the gels were transferred to nitrocellulose transfer membranes  by blotting  using a For mass spectrometric analyses, RP-HPLC fractions were analyzed by MALDI-TOF mass spectrometry. Each sample was loaded onto a MALDI plate with a matrix of α-cyano-4-hydroxycinnamic acid. Mass spectrometric analysis was carried out on a Bruker Daltonics Reflex IV instrument  equipped with a nitrogen laser, operating in positive mode. Each spectrum was accumulated for at least 200 laser shots and Bruker peptide calibration standards were used for calibration.Automated N-terminal amino acid sequence analysis was performed on a pulsed-liquid sequencer equipped with a PTH analyser .White lupin 11S globulin was purified from a mature dry seed protein extract, according to established protocols, based on anion exchange chromatography separation . In the 292-H296 and H319-H322 respectively, could be responsible for the protein’s metal binding capacity. By using an anti-his-tag antiserum, the reactive 11S globulin chains were shown on Western blots to be those of 42.1 and 40.4 kDa, indicated in From the white lupin 11S globulin amino acid sequence available in the databases  to make the two graphs comparable. Slopes were similar, thus suggesting a correlation between removal of the his-tag-like peptide region and loss of NiNTA-binding capacity of the lupin 11S globulin.Densitometric data, obtained by scanning the most representative his-tag band of 42.1 kDa in SDS-PAGE analyses of the treated samples during the course of incubation , were th288, R289 and H290. In parallel, N-terminal sequencing of the peptide confirmed the cleavages in the indicated positions starting from RRHP . The peaks were collected and analyzed by MALDI-TOF spectrometry. Only the major peak gave reliable molecular masses around 3,000 Dalton . These mrom RRHP . This lain vitro degradation, proteins were extracted directly from germinating cotyledons with sample denaturing buffer, at 100°C and then analyzed by SDS-PAGE under reducing conditions. The separated polypeptides were blotted onto nitrocellulose membrane and reacted with anti-his-tag antibodies. The results are shown in in vitro incubation of the lupin 11S globulin with the -R-R- endopeptidase, though with expectedly different dynamics. It is worth noting that one of the two anti-his-tag reacting polypeptides present in the mature quiescent seeds  was carried out on 9 polypeptide sequences, namely lupin 11S globulin (UniProtKB:Q53I54), pea legumin (UniProtKB:P02857), cruciferin A chain (PDB: 3KGL) and the 6 cruciferin sequences from 3KGL which were fixed in the crystallographic structure [Accelrys). The results of this alignment (not presented) were highly comparable with that of Tandang-Silvas et al. [The release of the his-tag-like region promoted by the -R-R- specific endopeptidase and the abundance of acidic and basic amino acids and histidine residues in the cleaved fragment suggest that this region and the target peptide bonds lie in an exposed peptide loop near the surface of the 11S globulin. The comprehensive structural comparisons of 11S globulin 3D structures published by Tandang-Silvas et al.  has allotructure . The diss et al. , with thin vitro with the -R-R- cleaving endopeptidase [In a previous work , the hypeptidase . No furtThe presence of independently acting regions of seed proteins has already, though not frequently, been documented, such as the case of antimicrobial peptides originating from seed storage protein hydrolysis, with activity against fungi and insects . In soybMany seed storage proteins contain from 1 up to 12 twin arginine motifs  and the ++-binding polypeptide, termed microplusin, has unequivocally been attributed to its copper depletion mode of action [Cucurbita maxima provicilin [Heliantus annuus proalbumin [This work did not aim to measure any physiologically-relevant activity of the liberated peptides; however their metal binding capacity speaks in favour of a metal mobilization/chelation role. Indeed, although little is known about the potentially limiting role of mineral nutrients in early metabolic and developmental processes during germination, our findings support an involvement of these metal-containing peptides in the reallocation of several ions from germinating cotyledons to root and hypocotyl tissues . A defenf action . The preovicilin  and Helioalbumin .This work, by showing the specific removal of the his-tag-like region of germinating lupin seed 11S globulin prior to globulin collapse, further supports the former hypotheses on an orderly deconstruction of storage proteins during germination rather than an extensive, random proteolytic digestion , 6 and aThe extent to which storage proteins lose intact peptides and whether the removed peptides have biological activities useful for the plants early growth are questions alluded to here and are worthy of further study. Nonetheless, the present findings on a natural metal chelator originating from a lupin storage globulin, through a mammalian-like specific proteolytic event, open up new perspectives in the identification of cryptic bioactivities related to seed ontogenesis and protection.


Toxoplasma gondii antibodies: a group of 55 toxoplasma-positive persons (TP) and a group of 46 toxoplasma-negative persons (TN). Absolute counts of several lymphocyte subsets decreased in the TP group, namely, T cells (p = 0.007), B cells (p = 0.002), NK cells (p = 0.009), CD4 T cells (p = 0.028), and CD8 T cells (p = 0.004). On the other hand, the percentage of CD8 T cells expressing CD38 and HLA-DR significantly increased during the follow-up in the TP group  as well as the intensity of CD38 and HLA-DR expression (MFI) on CD8 T cells . In the TN group, analysis of the kinetics of immunological parameters revealed no significant changes over time. In conclusion, the results suggest that latent T. gondii infection modulates the immune response during HIV infection.A relationship between latent toxoplasmosis and the immune system during HIV disease is poorly understood. Therefore, the aim of this follow-up study was to characterize immunological parameters in HIV-infected patients with latent toxoplasmosis and noninfected individuals. A total of 101 HIV-infected patients were enrolled in the study. The patients were classified into two groups based on anti- Toxoplasma gondii is an intracellular protozoon causing one of the most prevalent infections. Apart from humans, T. gondii infects various mammals and birds, with small felids playing a major role as definitive hosts of the parasite. Humans are infected either by consumption of undercooked meat containing a tissue cyst or by ingestion of oocysts shed by the definitive host in feces. In women infected during pregnancy T. gondii infection can lead to vertical transmission and fetal infection [nfection , 2. T. gondii infection may be reactivated as a result of immune disorders [The primary infection in immunocompetent individuals is mostly asymptomatic or can be manifested as lymphadenopathy and is usually followed by a lifelong latent infection. However, from this state of latencyisorders . Despiteisorders , 5. T. gondii different immune cells were shown to have a role in host resistance to this organism [γ and interleukin- (IL-)12. Long-term host resistance is provided by T and B cells. However, the mechanisms of immune surveillance are not fully understood. Regarding the influence of latent toxoplasmosis on humans, several effects have been documented. Latent toxoplasmosis increases the risks of Parkinson's disease [ T. gondii infection may lead to immune suppression in both mice and humans [Following infection withorganism . T cells disease , behavio disease –10, or a disease , 12. Mord humans , 14. DesTherefore, the aim of the study was to characterize and compare different immunological parameters, including expression of activation markers on CD8 T cells of HIV-infected patients with latent toxoplasmosis and HIV-infected individuals without latent toxoplasmosis during a one-year follow-up.T. gondii antibodies, the patients were classified into two groups: a group of toxoplasma-positive (TP), which comprised 55 seropositive patients, and a group of 46 toxoplasma-negatives (TN). In A total of 101 HIV-positive patients  registered at the AIDS Centre at Na Bulovce Hospital in Prague were enrolled in the study between December 2012 and April 2013. This prospective study was conducted in accordance with the Helsinki Declaration as revised in 2000 after obtaining the approval from the local ethics committee. A written informed consent was obtained from all study participants. The patients were randomly selected in order to represent the entire cohort of HIV patients treated at the centre. Based on anti-Routine immunophenotyping  and an analysis of the surface expression of activation markers (CD38 and HLA-DR) on CD8 T cells were done from EDTA-treated peripheral blood using monoclonal antibodies (Ab)  . AnalysiT. gondii antibodies [During the follow-up, the complement-fixation test (CFT) and IgM/IgG ELISA test were used for detection of anti-tibodies , 18. Post-test. The comparison of the change magnitude between TP and TN group was performed using an analysis of variance with the interaction term between the group and time. The tests were applied to logarithmically transformed data. All statistical tests were evaluated as two-sided at a significance level of 0.05. Stata release 9.2  statistical software was used for statistical analysis.Geometric means together with corresponding 95% confidence intervals (95% CI) were calculated to characterize the location and variability of the analyzed variables in the groups. The analysis of mean change during follow-up in individual groups was based on Student's paired  T. gondii and seroconverted. The comparison of laboratory parameters between the TN and TP groups at the time of enrollment and after a one-year follow-up is presented in p = 0.007), B cells (p = 0.002), NK cells (p = 0.009), CD4 T cells (p = 0.028), and CD8 T cells (p = 0.004). On the other hand, as shown also in Figures p = 0.003, p = 0.042, resp.) similar to the intensity of CD38 and HLA-DR expression (MFI) on CD8 T cells . In the TN group, the analysis of the kinetics of immunological parameters revealed no significant changes over time.During the study follow-up, none of the 46 initially TN patients were infected with T. gondii in comparison with TN individuals during a one-year follow-up. Latent toxoplasmosis status was defined as the presence of anti-T. gondii antibodies without any clinical symptoms. During the study, in none of the TP patients latent toxoplasmosis had been reactivated. This finding reflects the fact that the majority of patients had good immunological parameters and only a few individuals equally distributed in both groups were enrolled with HIV infection in stage 3 (AIDS). Moreover, the incidence of reactivation is quite low and was documented to be 3.4 cases per 1000 anti-T. gondii positive HIV-infected patients per year in the Czech Republic [T. gondii specific CD4 and CD8 T cell responses [ T. gondii infection on HIV disease progression and immune parameters is not clear.In this prospective follow-up study, we characterized the immune response in HIV-infected patients latently infected withesponses . Howeveresponses , 21. Des T. gondii infection. This finding is in line with a previous study in HIV-negative persons, where male patients with latent toxoplasmosis had lower B cells, NK cells, and monocytes [ T. gondii infection is associated with the decline of this T cell subset [3 in both groups during the follow-up period, and the mean decrease in the TP group was 56 cells/mm3 compared to 36 cells/mm3 in the TN group. The observed decline of CD4 cell count can be attributed to HIV progression but partly also to the potential effect of latent toxoplasmosis. In a previous study of HIV-negative individuals CD4 cell count was not influenced by latent toxoplasmosis, but the discrepancy may also reflect the fact that the enrolled HIV-negative patients had different immunopathological conditions [We observed the decrease in the majority of lymphocyte subset counts in the group of HIV-infected patients with latentonocytes . CD4 T cl subset . In our nditions . T. gondii as important producer of IFN-γ [ T. gondii infection [γ production and protective immunity against T. gondii infection [ T. gondii latent infection may have had an influence on a more rapid long-term progression of HIV disease.Although CD4 T cells play a key role in the immunity againstof IFN-γ , CD8 T cof IFN-γ , 24. In nfection . Specifinfection . Similarnfection . The treIn our study, the percentages of activated CD8 T cells characterized by the expression of CD38 and HLA-DR increased in the TP group during the follow-up. Similar to the lymphocyte subset counts, these changes were not observed in TN patients. Previous studies demonstrated that increased expression of CD38 and HLA-DR markers on T cells correlates with disease progression and depletion of CD4 T cells, better than the level of the viral load . Also, m T. gondii infection in modulation of the immune responses during HIV infection. The results suggest that latent toxoplasmosis has an effect on the kinetics of lymphocyte subset counts and surface expression of immune activation markers in HIV-infected persons. The observed changes are mild and further studies are warranted to elucidate the impact of highly prevalent T. gondii infection on the complex immunopathogenesis of HIV infection.This study presents new findings about the role of latent


The rapid, sensitive and low-cost detection of macromolecular biomarkers is critical in clinical diagnostics, environmental monitoring, research, etc. Conventional assay methods usually require bulky, expensive and designated instruments and relative long assay time. For hospitals and laboratories that lack immediate access to analytical instruments, fast and low-cost assay methods for the detection of macromolecular biomarkers are urgently needed. In this work, we developed a versatile microparticle (MP)-based immunoaggregation method for the detection and quantification of macromolecular biomarkers. Antibodies (Abs) were firstly conjugated to MP through streptavidin-biotin interaction; the addition of macromolecular biomarkers caused the aggregation of Ab-MPs, which were subsequently detected by an optical microscope or optical particle sizer. The invisible nanometer-scale macromolecular biomarkers caused detectable change of micrometer-scale particle size distributions. Goat anti-rabbit immunoglobulin and human ferritin were used as model biomarkers to demonstrate MP-based immunoaggregation assay in PBS and 10% FBS to mimic real biomarker assay in the complex medium. It was found that both the number ratio and the volume ratio of Ab-MP aggregates caused by biomarker to all particles were directly correlated to the biomarker concentration. In addition, we found that the detection range could be tuned by adjusting the Ab-MP concentration. We envision that this novel MP-based immunoaggregation assay can be combined with multiple detection methods to detect and quantify macromolecular biomarkers at the nanogram per milliliter level. The quantitative detection of biomarker(s) is very important in clinical diagnostics , 2 envirThe objective of this work was to develop a sensitive, low cost and versatile microparticle (MP)-based immunoaggregation assay, for the quantitative and qualitative detection of macromolecular biomarkers. Streptavidin–functionalized Microparticle (MP) (Dynabeads M-280 with a diameter of 2.8 μm), biotinylated polyclonal rabbit anti-goat IgG (rAb) and goat anti-rabbit IgG (goat IgG) (labeled with Alexa Fluor 488) were bought from Life Technologies . Goat anti-human ferritin polyclonal antibody (gAb) and human ferritin were purchased from United States Biological . NHS-Fluorescein, NHS-PEG4-Biotinyltion and Zeba spin desalting column were purchased from Thermo Scientific . Dimethyl sulfoxide (HPLC grade) was bought from Alfa Aesar (USA). Phosphate buffer saline , and bovine serum albumin (BSA) were obtained from Sigma-Aldrich .To prepare the immunoaggregation sample, MP and biotinylated rAb were diluted to 0.16 mg/mL and 6.4 ng/mL separately in PBS containing 0.1% BSA. Equal volumes of 166.7 μL of diluted MP solution and 166.7 μL of diluted rAb solution were mixed for 30 minutes on a thermo mixer agitated at 650 rpm at room temperature. Biotinylated rabbit anti-goat Abs were conjugated to MP to form rAb-MP through the streptavidin-biotin binding. The conjugated solution was placed on a magnet to separate rAb-MPs from the solution and the unconjugated Ab supernatant was discarded. The rAb-MPs were resuspended with PBS with 0.1% BSA to the concentrations of 53.4 μg/mL. Different concentrations of goat IgG, which was used as model biomarker, were prepared with a range from 0.1 ng/mL to 320 ng/mL. 333.4 μL of Ab-MP solution was mixed with 166.7 μL of goat IgG solutions at different concentrations for 30 min on a thermal mixer at 650 rpm at room temperature. The same procedure was used for human ferritin detection. Goat anti-human ferritin polyclonal antibody (gAb) functionalized MP (gAb-MP) were suspended in PBS with 0.1% BSA to two different concentrations, 53.40 μg/mL and 213.4 μg/mL. The concentration of human ferritin ranged from 0.1 ng/mL to 416 ng/mL in PBS with 0.1% BSA. In a parallel study, 10% fetal bovine serum  was used to dilute the human ferritin to different concentrations ranging from 0.1 ng/mL to 416 ng/mL to mimic the biomarker detection in the complex medium.fn) was defined as the ratio of the number of individual particles in aggregates to the number of all individual particles. The same characterization methods were used for the human ferritin detection. To confirm the result, Ab-MP aggregates were diluted to 100 mL PBS with 0.1 mg/mL BSA and the size and counts of the particles in the sample were measured using a particle counter with a detection range of 0.5∼500 μm . The volume fraction of aggregates (fv) was calculated as volume ratio of large particles (3.0 μm∼10 μm) to all particles (1.5 μm ∼10 μm).To characterize the immunoaggregation, rAb-MP solutions with and without biomarker (goat IgG) were imaged with a fluorescent microscope  under a 40X objective lens though bright field filter and GFP filter (494/518 nm) respectively and analyzed with MetaMorph microscopy automation & image analysis software . The rAb-MP aggregates solutions formed under different goat IgG concentrations were dropped to a glass slide and covered with a cover slip. The number of rAb-MP, 2-rAb-MP aggregates, 3-rAb-MP aggregates and 4-rAb-MP aggregates were counted separately through the Olympus IX-81 fluorescent microscope under the bright field mode. To ensure accuracy and repeatability, more than 1000 particles were counted for each sample and 3 samples were prepared and tested for each goat IgG concentration. The number fraction of aggregates  of rAb-MP aggregates were achieved at 40 ng/mL of goat IgG. Within the range from 0.1 ng/mL to 40 ng/mL, the volume fraction of aggregates went up with the increase of goat IgG concentration, while it went down with the increase of goat IgG concentration above the turning point (40 ng/mL). This is because goat IgG at high concentrations saturated rAb on MPs; therefore the number of unreacted rAb on MP was too low to aggregate.Quantitative detection of biomarker is needed for many applications. We hypothesized that at the given particle concentration, the number or volume ratio of aggregates to total particles is proportional to the antigen concentration. To test our hypothesis, the immunoaggregation behaviour of rAb-MP as a function of goat IgG concentration in PBS solution was measured by using the IX-81 microscope under the bright field mode. The concentration of rAb-MP was kept constant at 53.4 μg/mL and mixed with different concentrations of goat IgG ranging from 0.1 ng/mL to 320 ng/mL. Single rAb-MP and 2-rAb-MP aggregates, 3-rAb-MP aggregates and 4-rAb-MP aggregates for each goat IgG concentrations were recognized and counted separately through recorded microscope images. The aggregates formed by more than 4 rAb-MPs were neglected in the calculation, since they were less than 1%. To evaluate the nonspecific aggregates, the negative control sample of rAb-MP in PBS without goat IgG was also counted; the number fraction of nonspecific rAb-MP aggregates was 6.8 ± 0.01%. The number fraction of rAb-MP aggregates for each goat IgG concentration was calculated by subtracting the nonspecific aggregates volume fraction value (6.8%) and had a clear relationship with goat IgG concentration as shown in fv) of gAb-MP aggregates (3.0 μm∼10 μm) to all particles (1.5 μm ∼10 μm) were calculated and were plotted versus ferritin concentration (fv) of larger particles to all particles more directly reflects the aggregation behavior of gAb-MP in this case. To ensure repeatability, at each ferritin concentration, 5 samples were prepared and measured. fv = 23.2%) of gAb-MP aggregates was achieved at the ferritin concentration of 41.6 ng/mL. Within the range from 1.04 ng/mL to 41.6 ng/mL, the volume fraction of gAb-MP aggregates to all the particles follows an upward trend with the increase of ferritin concentration. At higher ferritin concentrations (>41.6 ng/mL), the volume fraction of gAb-MP aggregates decreased with the increase of ferritin concentration.Human ferritin was used as a real biomarker to validate whether MP-based immunoaggregation assay could be applied for the real human biomarker detection. Ferritins exist in many organisms, including vertebrates, invertebrates, plants, fungi, and bacteria, and they function as iron storage proteins . The abnntration . Since tfn) of nonspecific aggregates of the negative control were 6.8 ± 0.3% and 6.8 ± 0.1% for gAb-MP concentrations of 53.4 μg/mL and 213.4 μg/mL respectively. The low number fractions of the nonspecific gAb-MP aggregation suggest that gAb-MPs were stable in 10% FBS. The number fraction of aggregates of each sample was obtained by subtracting the number fraction value of the nonspecific aggregates. Detection of biomarkers is a challenge at low concentrations in complex media, such as body fluid, blood, urine, etc., since the nonspecific binding of biomolecules to the capture probes or sensing surfaces may cause the false positive result and decrease the detection sensitivity , 20. To Since different biomarkers exist at different concentrations, it is highly desired that the detection range of the assay be adjustable to match the various concentrations of different biomarkers. Based on the immunoaggregation principle, the detection range can be tuned by changing the Ab-MP concentration. We think this detection method will be particularly useful for hospitals or laboratories that need rapid clinical detection but lack immediate access to analytical instruments. Since the capture probe, Ab-MP conjugates could be prepared before the detection, the assay time for this method is less than 1 hour, which is shorter compared to the conventional ELISA method. Furthermore, the quantitative or qualitative detection of the biomarker can be conducted using optical microscopes with a hemocytometer, which are inexpensive and usually available in hospitals and biological labs. If we add equal amount of particles to different samples with the same volume, the concentration of total particles in all samples will be the same. Since the volume of the fluid in the hemocytometer is fixed, the number of the aggregates in the hemocytometer can be directly linked to the antigen concentration. Therefore, we only need to determine the number of aggregates instead of recording both the counts of aggregates and individual particles. Moreover, the software for counting the particles and aggregates, such as Image J, could be used to replace manual counting to further reduce the assay time. It is expected that complex samples, such as blood and body fluid, may cause higher non-specific aggregations that will increase the noise and detection limit. However, the detection limit of for our method is 0.1 ng/mL, which is lower than that of commercial available human ferritin ELISA kits (approximately 1 ng/mL), so the blood sample can be conveniently diluted to a suitable concentration before detection. On the other hand, as we demonstrated, the detection range can be tuned by adjusting the Ab-MP concentration. For concentrated blood sample, a lower Ab-MP concentration can be used to achieve a lower detection range. The same method can be used to detect multiple biomarkers simultaneously. Microparticles with different color and capture probes can be premixed and then added to the sample. Each type of the biomarker will cause the aggregation of microparticles with the specific color. If the sample contains different types of biomarkers, aggregates with different color can be detected. For the rapid qualitative detection, given the fixed sample volume and particle concentration, the threshold value of aggregates can be predetermined and sample can be considered as the positive once the number of aggregates is over the threshold value.An innovative MP-based immunoaggregation assay for macromolecular biomarker detection was demonstrated. The biomarker can cause the aggregation of Ab functionalized microparticles and the number or volume fraction of aggregates increased with the concentration of biomarker among the detection range. The detection range is 0.1 ng/mL to 40 ng/mL and 0.1 ng/mL to 208 ng/mL for two model biomarkers, goat IgG and human ferritin, respectively. The detection range can be further extended by adjusting the Ab-MP concentration. It has also been proved that the MP-based immunoaggregation assay can be applied to real biomarker in 10% FBS, which is close to the real detection environment. To the best of our knowledge, it is for the first time that the immunoaggregation method was reported for biomarker detection. This novel MP-based aggregation immunoassay could potentially be used to detect macromolecular biomarkers at the ultra-low concentration (nanogram per milliliter) without using expensive and dedicated analytical instruments.


ZC3HC1, which encodes NIPA. In this study, we show that rs11556924 polymorphism is associated with lower regulatory phosphorylation of NIPA in the risk variant, resulting in NIPA with higher activity. Using a genome-editing approach we show that this causes an effective decrease in cyclin-B1 stability in the nucleus, thereby slowing its nuclear accumulation. By perturbing the rate of nuclear cyclin-B1 accumulation, rs11556924 alters the regulation of mitotic progression resulting in an extended mitosis. This study shows that the CAD-associated coding polymorphism in ZC3HC1 alters the dynamics of cell-cycle regulation by NIPA.Genome-wide association studies have to date identified multiple coronary artery disease (CAD)-associated loci; however, for most of these loci the mechanism by which they affect CAD risk is unclear. The CAD-associated locus 7q32.2 is unusual in that the lead variant, rs11556924, is not in strong linkage disequilibrium with any other variant and introduces a coding change in  Coronary artery disease (CAD)Genome-wide association studies (GWAS) have identified more than 56 loci, which contribute to risk of CAD –7. Many –et al.  protein encoded by ZC3HC1 . Ser-395 is also in the same region of the protein, although a bit further away in the secondary structure. Given these findings we hypothesized that the Arg-363-His polymorphism in NIPA affects Cyclin-B1 dynamics and cell cycle regulation through an impact on phosphorylation of NIPA and undertook experiments to investigate this. Our study provides a potential functional basis for the effect of allelic variation at rs11556924 on altered CAD risk.The function of NIPA itself is regulated by phosphorylation at key residues, Ser-354 and Ser-359 targeted by the ERK1/2 kinases  and Ser-Fig. 1B). In our model, in the Arg-363 (CAD-associated) variant, the arginine side chain forms a strong hydrogen bond with the Ser-395 residue, the phosphorylation target of CDK1. In the His-363 form of the protein, however, the side chain of the histidine residue points toward the inner core of the leucine rich repeat binding domain and does not interact with Ser-395. There are also conformational changes in the leucine-rich repeat protein binding domain. In particular, the His-363 variant has a looser structure due to the looser winding of several α-helices.To determine if the Arg-363-His polymorphism has the potential to impact on phosphorylation of NIPA, we generated a predicted structural model of the two forms of the protein B. In ourin vitro kinase assays. To achieve this, the 2 NIPA variants, tagged with MBP , were bacterially expressed and then used as substrates for a kinase assay using recombinant CDK1 and ERK2 kinases. MBP alone was used as a negative control and was not phosphorylated. A kinase assay using CDK1 kinase showed that phosphorylation of NIPA occurred at a mean rate of 0.494 ± 0.044 pmol phosphate/min in the Arg-363 variant compared with 0.694 ± 0.141 pmol phosphate/min in the His-363 variant, so phosphorylation is occurring significantly slower in the CAD-risk variant of the protein (p = 0.002) (C). Phosphorylation by ERK2, however, showed no significant difference between phosphorylation of the 2 NIPA variants (p = 0.622), with a mean rate of 0.184 ± 0.065 pmol phosphate/min in the His-363 variant and 0.198 ± 0.025 pmol phosphate/min in the Arg-363 (D).To experimentally confirm whether these changes in the structure of the protein are sufficient to alter the phosphorylation of the protein in this region, we used = 0.002) C. Phosph Arg-363 D.2 beads and used SILAC quantitation to look for differences in the level of phosphorylation at these sites. A total of 39 phosphorylation sites in NIPA were identified in our phospho-enriched samples. We were able to quantify the relative level of phosphorylation at 30 of these sites. Phosphorylation at Ser-395 was measurable using this approach, but phosphorylation at Ser-354 and Ser-359 was not, due to low levels of peptide detection. The site at Ser-395 had 44% lower phosphorylation of the Arg-363 variant compared with the His-363 variant (E), suggesting that the polymorphism is perturbing phosphorylation at this site.To further validate this effect of rs11556924 on the regulatory phosphorylation of NIPA, we used MS/MS proteomics to carry out phospho-analysis of the NIPA protein. To achieve this, vectors expressing each variant were generated and expressed in HEK293T cells. We then immunoprecipitated NIPA and used MS/MS to examine its phosphorylation. We enriched for phospho-peptides using TiO variant E, suggesA). rAAV is an effective method for generating small genetic alterations . Examining multiple lines of each genotype helps to ensure that differences between lines of different genotypes are down to the changes generated at rs11556924 and not due to off-target effects.Having shown that the coding variation caused by rs11556924 alters regulatory phosphorylation of NIPA, we next investigated the effect of the SNP on endogenous protein function in cells. To do this we created isogenic lines differing only at rs11556924, using a recombinant Adeno-Associated Virus (rAAV) genome editing approach A. rAAV ierations , 23. rAAerations . As the  editing , 24–27. iting 24–B. ExaminKLHDC10 gene, which is the next gene downstream from ZC3HC1 (19kb away)  (C) or KLHDC10 (p = 0.291) (D), indicating that the SNP does not alter expression of either of these genes.A previous study had suggested that rs11556924 may be associated with expression of the kb away) . To test= 0.442) C or KLHD= 0.291) D, indicaPhosphorylation of NIPA at Ser-395 acts to deactivate NIPA, preventing it from ubiquitinating Cyclin-B1 and thus allowing accumulation of Cyclin-B1 in the nucleus. We therefore next studied the rs11556924 genome edited lines to test whether the SNP genotype impacted on Cyclin-B1 stability.A). The overall trend was significant (ANOVA p = 0.017) and post-hoc tests showed a significant difference between homozygotes (p = 0.015). This finding was contrary to our expectations, a reduction of NIPA phosphorylation in the CAD risk genotype group would have been expected to result in more active NIPA and consequently lower levels of Cyclin-B1. To further examine the impact of rs11556924 on Cyclin-B1 regulation we needed to examine the impact of rs11556924 on its stability in the nucleus more directly.We first examined total Cyclin-B1 in cells of the three genotypes using Western blotting with an α-Cyclin-B1 antibody. We found that Cyclin-B1 levels were higher in CAD-risk genotype cells compared with CAD-non-risk cells, with the heterozygote intermediate between the homozygotes A. The ovB, i). We then quantified the relative amount of Cyclin-B1-GFP in the nucleus at 2 min intervals in the time around mitotic entry but prior to nuclear-envelope breakdown. The rate of nuclear entry of Cyclin-B1-GFP was reduced in cells of the risk genotype with risk cells having a mean rate of 0.095 ± 0.032 compared with 0.134 ± 0.048 in cells of the non-risk genotype (p = 0.004) .To investigate the effect of rs11556924 on the nuclear stability of Cyclin-B1, we examined the effect of genotype on Cyclin-B1 at the time in the cell cycle when this effect is likely to be most important, the time of mitotic entry as cyclin-B1 begins to accumulate in the nucleus. We hypothesized that the rate of nuclear accumulation could be altered, perturbing cell cycle dynamics. To test this hypothesis, we expressed Cyclin-B1-GFP in the genome edited cell lines, and used live microscopy to monitor the nuclear entry of Cyclin-B1-GFP B, i. We = 0.004) B, ii.C). There was a trend toward a lower amount remaining in the CAD-risk genotype cells compared with CAD-non-risk, although this difference was not significant (p = 0.167).To confirm that the effect of rs11556924 on Cyclin-B1 nuclear accumulation was due to altered stability of Cyclin-B1, we tested the effect of rs11556924 on the stability of endogenous Cyclin-B1. To achieve this, we blocked protein synthesis in our genome edited cell lines using cycloheximide. Total Cyclin-B1 levels were determined by Western blotting analysis in cells treated with cycloheximide for 5 h and then the relative amount of Cyclin-B1 quantified C. There i.e. the proportion of cells in mitosis at any one time. We found an increase in the mitotic index of the cells of the CAD-risk genotype, with a mean of 5.50 ± 0.26% compared with CAD-non-risk cells with a mean of 3.62 ± 0.30% with the heterozygote again showing an intermediate phenotype (A) (ANOVA p = 0.012). Post-hoc testing showed a significant difference between homozygotes (p = 0.010).Having demonstrated that rs11556924 alters the ability of NIPA to regulate the levels of Cyclin-B1 in the nucleus, we next investigated the effect of this altered regulation on control of the cell cycle. Initially, we tested the effect of rs11556924 genotype in the genome-edited cell lines on the mitotic index of these cells, henotype A  (B).Given our observation that cells of the risk genotype accumulate Cyclin-B1 into the nucleus more slowly , our hyp= 0.011) B.p = 0.652) (C).To test whether perturbation of Cyclin-B1 dynamics resulted in errors in mitosis, which could in turn result in a longer mitosis, we assessed the success of chromosome segregation by carrying out chromosome spreads of metaphase cells and counting the number of chromosomes in each cell. Errors in chromosome segregation would be expected to cause a widening in the distribution of chromosome number in the cells. We did not see this effect; the variation in chromosome number was the same in cells of the non-risk and risk genotypes (= 0.652) C.p = 0.522) (D).As the dynamics of Cyclin-B1 accumulation and the duration of mitosis are perturbed by rs11556924, this would be expected to alter the rate of proliferation of the cells. In order to test the effect of the SNP on proliferation, we used a Sulforhodamine-B assay of cell number across 72 h of cell growth in the genome-edited lines. There was no difference between cells of the different genotypes (= 0.522) D.p = 0.251) (E).Given that in the CAD-risk genotype cells, mitosis is taking longer, but these cells do not proliferate more slowly, we wondered whether this apparent contradiction was caused by decreased apoptosis in the CAD-risk cells. To test this, we measured apoptosis in the cell lines using a flow cytometry annexin-V assay. We found no detectable difference in the levels of apoptosis between cells of different rs11556924 genotypes (= 0.251) E.GWAS studies have been very fruitful in identifying SNPs that are associated with altered risk of various complex diseases, including CAD. In many cases, there are several variants in high linkage disequilibrium at each locus that are associated with disease and identifying the causal SNP can be challenging . As a reZC3HC1 gene has been shown to be associated with CAD, with a 9% increased risk with each additional copy of the CAD-associated allele . Consequently, NIPA activity is prolonged in cells carrying the CAD-risk genotype, causing slower accumulation of Cyclin-B1 in the nucleus of these cells (C) and an increase in total Cyclin-B1 (A), this is explained by the slowed progression through mitosis of cells of the CAD risk genotype, resulting in an increased mitotic index (A). As there are more cells in mitosis, the average level of Cyclin-B1 in the cells appears higher, although in fact it accumulates more slowly in the nucleus (B).We demonstrate that the rs11556924 arginine variant that is associated with CAD risk perturbs the function of NIPA, resulting in an alteration in Cyclin-B1 dynamics. Specifically, the CAD risk variant alters the rate of regulatory phosphorylation of NIPA by Cyclin-B1-CDK1, decreasing the level of NIPA phosphorylation in cells of the risk genotype C. Conseqse cells . This rese cells , 33. Altse cells C and an yclin-B1 A, this iic index A. As the nucleus B.ZC3HC1 on Cyclin B1 dynamics and the cell cycle could therefore influence the development and progression of CAD through effects on multiple cell types and at different stages of the chronic process of CAD. It should be emphasized that the DLD-1 cell line was used in these studies. Although the effect of the rs11556924 on NIPA function is likely to be generic, further investigation will be required to confirm these effects in CAD relevant cell types.The cell cycle is known to be important in the development of cardiovascular disease . This inB), considering that the entire cell cycle length is 27.7 h , it is not surprising that we did not observe a difference in proliferation. A difference of 3.4 min in a cell cycle of 27.7 h would correspond to a difference of 0.2% in the total cycle length. Our proliferation assay was across ∼3 cell cycles, so you would expect approximately a 0.6% in cell number at the end of the assay. Clearly, such a small difference would be extremely challenging to detect in a proliferation assay. Of course, in the context of coronary artery disease, even a subtle difference in cell cycle regulation could have a substantial impact on disease progression over the course of the months and years over which CAD develops.In the DLD-1 cells used in our study, the impact of rs11556924 on Cyclin-B1 dynamics and mitosis did not result in discernible differences in the rate of cellular proliferation between CAD-risk genotype and CAD-non-risk genotype cells. Our additional analysis suggests that this effect is not due to increased apoptosis in the non-risk genotype cells. There are several possible explanations for this. It is possible that under basal conditions the cells are able to compensate for this delay at other stages of the cell cycle and additional stimuli or stresses may be required to bring out the impact. It is most likely, however, that any difference in proliferation would be subtler than we are able to detect. Given the small difference between mitosis length in the non-risk and risk genotype cells  were then added using the program, Ionize, to ensure that the system was electrically neutral. This structure was then solvated in a box of water. The dimensions of the box were chosen to ensure the protein was surrounded by a boundary of at least 8 Å of water in all directions. The solvated system was then subjected to 50,000 cycles of MM energy minimization, before performing a 1-ns MD simulation. Starting from the structure generated from the previous MD simulation of the fully solvated wild-type, Arg-363 was mutated to His+-363. The mutant system was then energy minimized (MM) for 50,000 cycles before performing a 1 ns MD simulation.The I-Tasser structure prediction program  was usedZC3HC1 open reading frame (ORF) with a C-terminal FLAG tag in a pCMV-Entry vector was obtained from Origene (Rockville). A QuikChange site-directed mutagenesis kit  was then used to mutate the wild-type (CAD-risk) version to the mutant (CAD-non-risk) version of the gene. The two versions of the ORF were then subcloned into the pLEICS-10 vector, which contains an N-terminal MBP directly prior to the ORF, by The Protein Expression Laboratory at the University of Leicester .The wild-type ZC3HC1 were transformed into BL21 (DE3) competent cells. To express the protein, bacteria were culture overnight, then a low-density culture seeded; this was allowed to grow for 2 h, then expression induced by the addition of 1 mm IPTG to the culture. Cells were allowed to express for 4 h, then pelleted by centrifugation, the pellets frozen, then protein extracted in PBST. Amylose resin  was used to purify the proteins according to the manufacturer's instructions. Purified proteins were used as a substrate in a kinase reaction using a commercial kinase buffer , 200 μm ATP, 2 μCi ATP32 and 1 unit of kinase. The reactions were stopped using phosphoric acid, followed by liquid scintillation counting using Ultima Gold scintillation fluid  and a Tri-Carb scintillation counter (Perkin-Elmer).The vectors expressing MBP-tagged versions of l-arginine-HCl and l-Lysine-HCl, “Heavy” samples contained l-arginine-HCl (U-13C6) and l-lysine-2HCl  . Dialyzed fetal bovine serum (Sigma-Aldrich) was used to avoid amino acid contamination from the serum. Cells were lysed, and anti-FLAG M2 beads (Sigma-Aldrich) used to immunoprecipitate the protein using standard methods. Immunoprecipitated proteins were separated by SDS-PAGE, and the band corresponding to NIPA was excised. The band was then trypsinized, phospho-peptides purified using TiO2 beads, the unbound (non-phospho peptides) were kept and analyzed separately and subjected to MS/MS on an LTQ Orbitrap by the University of Leicester Proteomics Facility PNACL. Data were analyzed using MaxQuant (ver. 1.5.0.30)  . N-termiB). The loxP-flanked selection cassette was then removed using Cre recombinase to leave a single loxP site, the location of which was designed to be unlikely to be detrimental. Clones were then expanded from single cells, the removal of the selection cassette confirmed by PCR, then were genotyped again to ensure correct isolation of clonal lines. This resulted in the generation of cell lines that were isogenic other than at rs11556924.Recombinant AAV targeting vectors were produced for us by Horizon Discovery . rAAV viruses were generated following transfection of the appropriate targeting and helper vectors in HEK293T cells, and rAAV viruses purified using an AAV purification kit . AAV was titrated using qPCR. Wild-type DLD-1 cells were infected with the rAAV viruses carrying both alleles of rs11556924 to generate cells of all three genotypes. Following infection cells were selected for using the G418 resistance cassette included in the vector, and selected clonal populations expanded and genotyped by PCR and sequencing B. The loAs the loci that are identified from GWAS have additive effects on CAD risk, it would be expected that any differing phenotypes between risk and non-risk genotypes would be intermediate in the heterozygote, consistent with a dosage effect. Therefore, it is important to obtain heterozygote knock-in lines as well as the homozygotes, to be able to test this.RNA was extracted using an RNeasy miniprep kit (Sigma-Aldrich), reverse transcribed with Superscript III (Fisher Scientific), and qPCR carried out with SensiMix  on a Rotorgene Q qPCR machine .Cellular Cyclin-B1 levels were measured using Western blotting analysis, carried out using standard procedures on total cell lysates with an anti-Cyclin-B1 antibody raised in mouse (1:1000) (Santa Cruz Biotechnology) and an anti-mouse IgG from sheep  (Fisher Scientific).2 supply. An Andor iXonEM+ EMCCD DU 885 camera is attached to the microscope for image collection using NIS-Elements software . Mitosis measurements were on untreated cells. For Cyclin-B1-GFP imaging, cells were transfected with a vector expressing GFP-Cyclin-B1  and anti-phospho-Histone-H3 .Chromosome counts were performed by trypsinizing the cells, treating them with a hypotonic solution, and then spreading them onto a microscope slide. The DNA was then stained with DAPI, and imaged using an EVOS Fluorescent Microscope (Fisher Scientific). Images were analyzed using the FIJI release of ImageJ (ver. 1.50a) . Apoptost test was used to test for differences between genotypes. For chromosome number, an F-test was used to compare variances. All barcharts represent mean ± S.D. Significance levels: n.s., p > 0.05; *, p ≤ 0.05; **, p ≤ 0.01.Statistical analysis was carried out using R  and GrapP. D. J., M. A. K., N. J. S., and T. R. W. designed the experiments. P. D. J., M. A. K., M. G. N., and M. G. M. carried out experiments and analyzed data. A. J. B. carried out protein modelling. C. L. S. designed and generated rAAV vectors. P. D. J., N. J. S., and T. R. W. wrote the paper. All authors analyzed the results and approved the final version of the manuscript.


Metabolic Syndrome (MS) is prevalant in China, especially according to the pediatric obesity group. Based on the MS-CHN2012 definition for Chinese children and adolescents the need to explore and establish a convienent MS screening become imminent. This study aims to investigate the optimal cut-off values, compare the accuracy for the (TriGlycerides (TG) to High-Density Lipoprotein Cholesterol (HDL-C)) (TG/HDL-C) ratio and Homeostasis Model Assessment Insulin Resistance (HOMA-IR) indexs to identify Metabolic Syndrome in obese pediatric population in China.A total sample of 976 children  aged from 6–16 years underwent a medical assessment including a physical examination and investigations of total cholesterol, high-density lipoprotein, low-density lipoprotein, triglycerides, insulin, glucose, and oral glucose tolerance test to identify the components of Metabolic Syndrome. The validity and accuracy between TG/HDL-C ratio and HOMA-IR were compared by Receiver Operating Characteristics analysis (ROC).TG/HDL-C ratio achieved a larger ROC Area under Curve (AUC = 0.843) than HOMA-IR indexes  to screen for Metabolic Syndrome. The cut-off values for MS were: TG/HDL-C ratio > 1.25 , HOMA1-IR > 4.59  and HOMA2-IR > 2.76 . The results kept robust after stratified by gender, age group and pubertal stage.TG/HDL-C ratio was a better indicator than the HOMA-IR to screen for a positive diagnosis for MS. Furthermore, the TG/HDL-C ratio was superior to the HOMA-IR indexes even after the control of possible confusions from the gender, age group and puberty stage. TG/HDL-C ratio proved a better index than HOMA-IR in screening for MS in obese children and adolescents. TG/HDL-C ratio has a discriminatory power in detecting potential MS in the Chinese obese pediatric population. The prevalence of obesity has increased dramatically in children and adolescents as China is gradually taking its place as one of the world’s economic giants, it is becoming an important public health problem –4. MetabFor a long period anthropometric measurements had been recognized as the convenient indicators in the predicting MS –10. AfteThis was a cross-sectional study. Study quality was assessed according to the checklist of STARD (STAndards for the Reporting of Diagnostic accuracy studies). 1 069 Obese children and adolescents between 6 and 16 years old, of both genders , consecutively registered at the inpatient ward from our clinic, the Children’s Hospital of Zhejiang University School of Medicine– Hangzhou, in China, between May 2007 and June 2013, were invited to participate in the study. A total of 976 female286 male690) obese schoolchildren with complete record were eligibly included in the current study. the Age- and sex-specific Body Mass Index (BMI) percentiles, developed by the Working Group for Obesity in China, were used to classify participants as obese (BMI ≥ 95 %) , 28 for 6 male690Subjects’ height and weight were measured according to our standard protocol . BMI wasVenous blood samples were collected after an overnight (≥12 h) fast. Subjects also underwent an oral glucose tolerance test . The samples were centrifuged, aliquoted and immediately frozen for future analysis in blind of the clinical information. Blood samples were also analyzed for concentrations of plasma glucose, triglycerides (TG), total cholesterol (TC), high-density lipoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol (LDL-C) and insulin. Serum lipids (enzymatic methods) and plasma glucose (glucose oxidase method) were assayed using the Modular DPP automatic biochemistry analysis system . HDL-C and LDL-C were measured directly. Insulin was determined by chemiluminescent micro particle immunoassay , developed in the key Laboratory at the Children’s Hospital which had an inter-assay coefficients of variation of <9.0 % and no cross-reactivity to proinsulin (<0.05 %).http://www.dtu.ox.ac.uk/homacalculator/index.php.In this study the presence of pediatric Metabolic Syndrome (MS) was determined according to the MS-CHN2012 MS definition  for the U test. A sample of 26 from the MS group and 26 from the Non-MS group achieved 90 % power to detect a difference of 0.2 between a diagnostic test with a Receiver Operating Characteristic (ROC) Area Under the Curve (AUC) of 0.8, and alterative diagnostic test with an AUC of 0.6 using a two-sided Z-test at a significance level of 0.05, The correlation between the two diagnostic tests is assumed to be 0.6. Prevalence of individual metabolic abnormalities of different groups was compared using the Chi-square test or Fisher’s exact test as appropriate. A receiver operating characteristic (ROC) curve was generated for the total studied population. The areas under the ROC curve (AUC) were calculated to evaluate the accuracy of the indicators by nonparametric method. The greater the AUC, the greater the discriminatory power of them for MS. The optimal cut-off value was denoted by the value that had the acceptable sensitivity, specificity and the closest point to the upper left corner of the ROC curve, which is often selected as the best combination of true-positive rate and false-positive rate [P < 0.05 was defined significance.Data was reported as median (interquartile range), and comparisons were performed using Mann–Whitney ive rate . The Z s1 069 Obese children and adolescents of both genders  aged from 6-16years were registered in this study. A total of 42 subjects were excluded because they did not satisfy inclusion criteria . Other exclusions were twelve subjects diagnosed with early-onset type 2 diabetes mellitus, nine with distress during BP monitoring, twenty with missing data in clinical or laboratory records and ten who refused to participate. Finally 976 participants were included in the analysis datasets. According to the MS diagnosis, overall our study showed that around 25.8 % of the 976 children and adolescents analyzed presented the syndrome, which was more prevalent in larger than 10-year-age obese individuals, especially those at puberty stage. But no difference was found between genders  than either HOMA1-IR or HOMA2-IR. The cut-off values for MS were: TG/HDL-C ratio > 1.25 , HOMA1-IR > 4.59  and HOMA2-IR > 2.76 . After stratified by age group, puberty stage and sex, the cutoffs of HOMA1-IR changed from 3.58–5.74 while the cutoffs of HOMA2-IR fluctuated from 1.92–2.99. However the cutoffs of TG/HDL-C varied slightly from 1.21–1.53. The Overall AUC-ROC values for the prediction of MS were 0.640, 0.625, and 0.843 by HOMA1-IR, HOMA2-IR and TG/HDL-C respectively. Significant difference of the AUC-ROC values between HOMA-IR and TG/HDL-C was found with a higher sensitivity and specificity. When stratified by age group, gender and puberty stage the AUC-ROC values for the prediction by HOMA-IR were still lower than those by TG/HDL-C.p > 0.05).Figure The present study investigated the optimal cut-off values for TG/HDL-C ratio, and HOMA-IR indexes to identify MS in a pediatric obese population. It was also demonstrated that the TG/HDL-C ratio was a better indicator than the HOMA-IR to screen for a positive diagnosis for MS. Furthermore, it was verified that the TG/HDL-C ratio was superior to the HOMA-IR indexes even after the control of possible confusions from the gender, age group and puberty stage.Previous studies demonstrated that the HOMA-IR indexes were a good indicator in identifying insulin resistance and MS in children , 23 and The TG/HDL-C ratio’s optimal cut off value to screen for MS is reasonable in obese children as the definition of the MS is the high amount of abdominal fat plus two of the four components including the Triglyceride and HDL cholesterol. So there is a high probability to be diagnosed as having MS. However, the optimal ratio of Triglyceride and HDL cholesterol can make a significant discrimination  to MS, wOne limitation of our study might be a potential bias caused by inconsistent measurements of Triglyceride and HDL cholesterol, because only data from patients in only one center with obesity and concomitant diseases are included in this study. Other bias with regard to the study population from a cross-sectional study may also have occurred; therefore the result may lack direct causality. Methodological aspects, such as biochemical measurements are more difficult to standardize in several years and the study result was accomplished only in one center, may contribute to the possible bias. However, experienced pediatricians and team staff in cooperation can make sure to comply with standardized procedures in anthropometric parameter measurement, analytical methodology and lab workup, which should make results from different year data comparable.This study demonstrates that TG/HDL-C ratio for screening MS may be a better index than HOMA-IR in screening obese children and adolescents with pediatric MS. We suggest that the accessible, effective and methodologically simple assessment of TG/HDL-C ratio might be powerful in detecting the early stage of potential MS in Chinese obese children and adolescents although further longitudinal study is needed to confirm the result.


Dictyostelium discoideum undergoes sexual maturation and forms gametes under dark and humid conditions. It exhibits three mating types, type-I, -II, and -III, for the heterothallic mating system. Based on proteome analyses of the gamete membranes, we detected expression of two homologs of the plant fertilization protein HAP2-GCS1. When their coding genes were disrupted in type-I and type-II strains, sexual potency was completely lost, whereas disruption in the type-III strain did not affect mating behavior, suggesting that the latter acts as female in complex organisms. Our results demonstrate the highly conserved function of HAP2-GCS1 in gamete interactions and suggest the presence of additional allo-recognition mechanisms in D. discoideum gametes.Fertilization is a central event in sexual reproduction, and understanding its molecular mechanisms has both basic and applicative biological importance. Recent studies have uncovered the molecules that mediate this process in a variety of organisms, making it intriguing to consider conservation and evolution of the mechanisms of sexual reproduction across phyla. The social amoeba  •Dictyostelium discoideum gametes.Two HAP2-GCS1 homologs are expressed in •Both homologs are responsible for the sexual cell fusion.•One mating type (III) out of 3 is HAP2-GCS1-independent, corresponding to female. To obtain gamete-phase cells, the growth-phase cells on an A-medium plate, which were fusion-incompetent, (IC-cells) were transferred to Bonner's salt solution (BSS) containing K. aerogenes and cultured on a gyratory shaker at 120 rpm in darkness at 22 °C for 16 h. These cells were referred to as fusion-competent (FC) cells.Heterothallic strains of 2.2K. aerogenes suspension in BSS and cultured in an incubator at 22 °C under darkness. Macrocysts were observed after 4–7 days. For the cell fusion assay, FC cells of the two strains were mixed in BSS at 5×105 cells/ml and incubated on a gyratory shaker at 120 rpm. Following 30 min incubation, EDTA was added to a final concentration of 1 mM to stop further cell fusion, and the number of unfused cells was counted to obtain the fusion index corresponding to the percentage of cells that participated in fusion. Cell fusion indices normally exceed 60% for compatible strains.Cellular ability with respect to macrocyst formation and cell fusion was determined following standard procedures . For the2.3D. discoideum protein database downloaded from dictyBase  sequences  and the primers listed in rnlA .Total cellular RNA was extracted using RNeasy RNA Extraction Kit  and converted to cDNA by PrimeScript™ II High Fidelity RT-PCR Kit  using random primers. PCR amplification was carried out by 2.5bsR cassette composed of the act15 promoter, bsr gene, and act8 terminator. An over-expression vector was constructed by inserting a cDNA between the v18 promoter and act8 terminator of pTMV18  and a weakly homologous protein, DDB0308507. We reconfirmed the gene sequences and named the two proteins HgrA (HAP2-GCS1 related protein A) and HgrB for convenience. Gene names are hgrA and hgrB, respectively. The latter was similar to HAP2-GCS1 only in the motif region. Both of them have a signal peptide at the N-terminus, the HAP2-GCS1 motif in the central region, and a probable transmembrane region near the C-terminus, suggesting membrane localization with a major extracellular portion and a short intracellular tail (D. discoideum (hgrA (gene for HgrA) that cover different coding regions. While HgrA was highly homologous to HAP2-GCS1 proteins in other organisms, HgrB was limited to the dictyostelid lineage. Phylogenetic analysis of these two proteins suggested that they diverged before the initial radiation of the social amoebae and evolved independently (When the crude membrane fractions from FC-cells of KAX3 (type-I) were subjected to SDS-PAGE followed by LC–MS/MS analysis using lar tail A. Two gescoideum . Howeverendently B.hgrA and hgrB, but it was much lower in V12, hgrB amplification being almost undetectable. To see if structural variations affected the detection of peptides and transcripts in type-II and -III strains, we determined the genomic sequences of hgrA (hgrB in V12 and WS2162 (D. discoideum but not as clearly as reported in other organisms. It was not highly elevated in the gamete-phase cells either.HgrA-derived peptides were detected only in the FC-KAX3 (type-I) sample at a low level and undetectable in V12 (type-II) and WS2162 (type-III) C. Hits f of hgrA  and hgrBd WS2162 . It is l3.2D. discoideum mating, we disrupted hgrA and hgrB in KAX3 by homologous recombination  (data not shown). If hgrA cDNA was introduced to one of its null mutants, and expressed under the control of the V18 promoter, both cell fusion and macrocyst formation abilities were restored . However, the disruption of either gene in type-IIIc did not hamper macrocyst formation with type-I  A and useh type-I Cb. Matinc was rather unexpected from their trace expression in V12, a wild type tester strain of type-II  (hgrB expression in the hgrA-disruptant was indistinguishable from the parent (There could be a possibility that the remaining gene complemented the counterpart loss in case of type-III(MO2930) Ea but hge parent Ea, sugge4D. discoideum ortholog of HAP2-GCS1, HgrA, plays a critical role for zygote formation mating-type dependently. This was the first demonstration for involvement of HAP2-GCS1 in amoebozoa mating, strengthening the broad conservation of its function across phyla with deep root. Another finding of ours was that the more distant homolog, HgrB, was also indispensable for gamete fusion. Homology of this protein is limited to the HAP2-GCS1 motif region, and overall sequence is unique to dictyostelid lineage, supporting the essential function of this motif  and not requiring (type-III) for gamete fusion, analogous to male vs. female in plants or plus vs, minus in algae.Despite extensive diversification of the sexual reproduction system, the essential step is conserved; shuffling of genetic materials following membrane fusion between two separate cells. Therefore, analyses of conjugation mechanisms in simple isogamous organisms can well aid the understanding of those in complex systems. Even apparently specific traits of the former give insights into evolutionary aspects of fertilization. Here we reported both conserved and unique features of gamete interactions in the cellular slime mold. The is motif . FinallyD. discoideum mating is currently elusive; they may function either sequentially or in multi-component fusion machinery  . Thus, gamete-phase cells in D. discoideum seem less specialized than other organisms.Although GCS1 and HAP2 were named after their specificity to generative haploid cells , express/IC=2.0) , coincidc. In consideration that mating-type locus genes should eventually control genes responsible for specific mating behavior of each sex, a logical explanation for the difference would be that the downstream genes or regulatory elements have accumulated changes in the wild strains, while keeping the mating-type intact. Generation of gene knockouts in tester strains, especially in V12, will clarify some of the questions remain unsolved.The principal reason for our use of the mating-type congenic strains was to circumvent the low efficiency of transformation in non-axenic strains. In addition to this, their common genetic background, except for the mating-type locus, should be beneficial for analytical studies like comparative proteomics as described below. On the other hand, it should be pointed that we observed differences between tester and congenic strain sets in protein and gene expressions; expression of HgrB was more repressed in V12 (type-II) than in type-IIChlamydomonas (Tetrahymena (D. discoideum mating, which could explain the situation that the gametes having both HgrA/B and Hgr-P do not self-mate. Identification and characterization of the hypothetical recognition molecules and Hgr-P are next key issues to be challenged and will clarify questions and hypotheses mentioned above. Expecting that expression of those proteins are under the control of mat locus genes, we are performing comparative proteomic analyses of the gamete membranes from mat locus congenic strains (It was shown that HAP2-GCS1 acts for membrane fusion event after sex-specific cell adhesion in ydomonas  and Tetrrahymena . Based o strains , Fig. S3 strains , both wi5D. discoideum possess two HAP2-GCS1 homologs. While 2 out of 3 mating-types require both of them for sexual cell fusion, the third one does not, suggesting male-female differentiation in this amoebozoa.Heterothallic strains of


Hexagonal boron nitride (h-BN), a well-known dielectric material, may afford the necessary protection. In this research, we demonstrated the van der Waals epitaxy of h-BN nanosheets on mechanically exfoliated graphene by chemical vapor deposition, using borazine as the precursor to h-BN. The h-BN nanosheets had a triangular morphology on a narrow graphene belt but a polygonal morphology on a larger graphene film. The h-BN nanosheets on graphene were highly crystalline, except for various in-plane lattice orientations. Interestingly, the h-BN nanosheets preferred to grow on graphene than on SiO Graphene has attracted global research interests across a wide range of applications ,2. HowevAs an analogue of graphene, h-BN shows a minimal lattice mismatch with graphene of about 1.7%, yet has a wide band gap -8 and loRecently, chemical vapor deposition (CVD) synthesis of h-BN on Ni -16 or Cu3N3H6) could be a promising choice as it would produce BN and hydrogen, which are both environmentally friendly.It has been demonstrated that van der Waals epitaxy by catalyst-free CVD can be a promising route for the growth of topological heterostructures -22. More2/Si and tended to exhibit a triangular morphology when grown on a narrow graphene belt. The h-BN nanosheets grown on graphene were highly crystalline, albeit with various in-plane lattice orientations.In this research, the van der Waals epitaxy of h-BN nanosheets on mechanically exfoliated graphene by catalyst-free low-pressure CVD, using borazine as the precursor to h-BN, was demonstrated. The h-BN nanosheets preferred to grow on graphene rather than on SiO2/Si) wafers by mechanical exfoliation from highly oriented pyrolytic graphite . The h-BN precursor (borazine) was synthesized by the reaction between NaBH4 and (NH4)2SO4 and purified according to our previous reports [2/Si was first annealed for 60 min in an argon/hydrogen flow  of 180 sccm to remove pollutants remaining on the graphene after mechanical exfoliation. During the growth process, borazine, in a homemade bubbler, was introduced to the growth chamber by another Ar flow of 2 sccm, while the Ar/H2 flow remained unchanged. The typical growth time was 5 min, while the pressure was 10 to 100 Pa. After the growth process, the tube was rapidly cooled to room temperature.h-BN nanosheets were synthesized in a fused quartz tube with a diameter of 50 mm. Graphene was transferred onto silicon oxide/silicon .Raman spectroscopy was performed in a Thermo DXR with 532-nm laser excitation . Atomic force microscopy (AFM)  and scanning electron microscopy (SEM)  were used to observe the thickness and morphology of the h-BN nanosheets. X-ray photoelectron spectroscopy (XPS)  was conducted to analyze the chemical composition of the films. The h-BN nanosheets with the graphene substrate were transferred to transmission electron microscopy (TEM) grids for further characterization. Both morphology images and selected area electron diffraction (SAED) patterns of the h-BN nanosheets were obtained by field emission high-resolution transmission electron microscopy (HRTEM)  lattice plane parallel to the surface of the SiO2/Si wafer [Raman spectroscopy provided a useful means of gleaning information about the lattice vibration modes of graphene and h-BN. After being transferred to SiOSi wafer ,2.I2D/IG ratio of graphene was less than 0.5, indicating the multilayer structure of the graphene. Moreover, a weak D peak of graphene at 1,350 cm-1 was observed from the Raman spectra (Figure -1, indicating the E2g vibration mode of h-BN, which was consistent with the reported values [The existence of graphene was shown by Raman spectra in Figure d values ,13-19. Id values  and grapd values ,28, showAccording to previous reports , the gas2/Si, as shown in Figure 2/Si substrate, while the C peak arose from the presence of graphene. The binding energies of B1s and N1s from the XPS spectra were 191.0 and 398.5 eV, respectively, which were in good agreement with reported values [XPS was used to analyze the chemical composition of the h-BN/graphene on the surface of the SiOd values ,19,33,34d values ,36. ThesWe have pointed out the reason for the nucleation of the h-BN on graphene. In fact, the deposition of h-BN nanosheets on graphene was performed as instantaneous nucleation followed by three-dimensional growth in our catalyst-free CVD growth. Similar results of three-dimensional growth in certain situations have been proved by previous reports ,32. As d2/Si, atomic resolution HRTEM was used to study the crystalline structure of the aforementioned h-BN nanosheets on their respective graphene substrates. Figure After the h-BN nanosheets on graphene were transferred to TEM grids after the etching of SiO2/Si, which offered the promise of potential applications for the preparation of graphene/h-BN superlattice structures. The h-BN nanosheets on graphene had a high degree of crystallinity, except for various in-plane lattice orientations. The synthesis of h-BN nanosheets on multilayer graphene has been studied, and h-BN nanosheets on few-layer and even monolayer graphene will be synthesized in future work. This may satisfy certain application requirements for topological heterostructures and graphene-related electronic devices.In summary, we have demonstrated the van der Waals epitaxy of h-BN nanosheets on graphene by catalyst-free CVD, which may maintain the promising electronic characteristics of graphene. The h-BN nanosheets tended to have a triangular morphology on a narrow graphene belt, whereas they had a polygonal morphology on a much larger graphene film. The B/N ratio of the h-BN nanosheets on graphene was 1.01, indicative of an almost stoichiometric composition of h-BN. The h-BN nanosheets preferred to grow on graphene rather than on SiOThe authors declare that they have no competing interests.YS, CZ, BL, and XX designed the experiments, and YS carried out most of the experimental work and material characterizations. CZ and BL synthesized the borazine. YS, CZ, BL, GD, and XX discussed the results, and YS drafted the manuscript. All authors have read and approved the final manuscript.


Sitagliptin (SG) increases serum GLP-1 (Glucagon-like peptide-1) through inhibition of the hormone degradation. Resistant starch (RS) induces GLP-1 expression by stimulating L-cells in the intestine. Sitagliptin and resistant starch may have a synergistic interaction in the induction of GLP-1. This possibility was tested in current study in a mouse model of type 2 diabetes. Hyperglycemia was induced in the diet-induced obese mice by a signal injection of streptozotocin (STZ). Sitagliptin (0.4g/100g diet) was tested in the mice (n = 55) with dietary RS (HAM-RS2) at three dosages . Energy and glucose metabolism were monitored in the evaluation of synergistic activity, and GLP-1 activity was determined in the GLP-1 receptor knockout (KO) mice. In the wild type mice, body weight and adiposity were reduced by sitagliptin, which was enhanced by RS (28g). Serum GLP-1 was induced and energy expenditure was enhanced by sitagliptin. Fasting glucose, insulin, and leptin levels were decreased by sitagliptin. The sitagliptin effects were lost in the KO mice (n = 25) although induction of serum GLP-1 by sitagliptin was even stronger in KO mice. The data suggests that sitagliptin is able to reduce adiposity and insulin resistance through induction of energy expenditure. The effect of sitagliptin is partially enhanced by RS. GLP-1 receptor may regulate serum GLP-1 by facilitating the hormone clearance. The drug sitagliptin (SG), made by Merck & Co., LLC, was first marketed in the United States in 2006  for imprResistant starch (RS) is a type of dietary fiber that stimulates GLP-1 secretion in L-cells through fermentation in the large gut. Short chain fatty acids (SCFAs) are generated in the fermentation to stimulate L-cells for GLP-1 expression and secretion ,6. SupplWe hypothesize that sitagliptin and resistant starch may have a synergistic interaction in the induction of GLP-1. To test this possibility, we conducted two cohort studies in a mouse model of type 2 diabetes. In the first study, sitagliptin was tested with different doses of HAM-RS2 to determine the synergy. In the second study, the role of GLP-1 was tested in GLP-1 receptor KO mice.All study procedures were approved by the IACUC committee at the Pennington Biomedical Research Center. In the first study, fifty five (N = 55), 7-week-old male C57BL/6 (WT) mice were purchased from the JAX Laboratories , and housed individually in shoebox cages in a climate-controlled environment  with a 12:12 hour light-dark cycle illuminated at 7 AM. The mice were fed a semi-purified HFD (42% energy) in order to induce weight gain. This pre-study diet also contained a small amount of HAM-RS2 (3% weight) in an attempt to maintain proper fermentative microbial communities despite dietary fat content. Mice were given one injection of a low dose of streptozotocin  during the third week on the HFD in order to induce hyperglycemia by partial β-cell loss to establish the type 2 diabetes model.Animals were stratified according to fasting blood glucose (244 ± 4 mg/dL) and body composition (25–26% body fat) as determined by nuclear magnetic resonance (NMR) spectroscopy, which was assessed after three weeks post-STZ injection. The mice were divided into six groups after a total of six weeks on the pre-study HFD (i.e. three weeks after STZ injection), and treated for ten weeks with isocaloric diets  (n = 9–10) containing HAM-RS2. Sitagliptin was given at 0.4% weight of diet while HAM-RS2 was tested at three dosages: 0, 15, and 28% weight of diet. Prior to consumption by the rodents, powdered diets were formed into bars by mixing with water and freezing overnight in a -20°C freezer, allowing food to be placed in hoppers as opposed to cups or tins. Fasted BG and body composition were assessed at the end of the tenth week of treatment.In the second study, male GLP-1R KO mice were used. The mice were treated with sitagliptin and HAM-RS2 (28% weight of diet) (SG+RS). The KO mice were obtained from Dr. Drucker at the University of Toronto, Ontario, Canada . WT and Metabolic rate and food intake were assessed by OxyMax/Comprehensive Lab Animal Monitoring System  for three days following a three-day acclimation period during the seventh and eighth weeks of the experimental. The chamber was maintained at the same temperature range and light-dark cycle as for the shoebox cages. Body composition and fasted BG were assessed at the end of ninth week and at the beginning of the tenth week of treatment.Fasting blood glucose (BG) was tested using a glucometer . After overnight fasting, blood was drawn by either tail or submandibular bleeding using sterile 5 mm Goldenrod Animal Lancets .In the first study, plasma was used to determine the concentration of active GLP-1 using ALPCO Diagnostic’s GLP-1 ELISA (Active 7–36) kit . An EMD Millipore Multiplex MAP kit  was used to assess the concentrations of various hormones including active GLP-1, leptin, and insulin in the second study. The GLP-1 assay used in the first study required plasma, which was collected in tubes containing another DPP-4 inhibitor (Millipore) after thirty minutes after re-feeding following an overnight fast. Fasting plasma was used for GLP-1 and other hormone tests in the second study.Mice were sacrificed via decapitation and blood samples were collected in EDTA tubes. The blood was centrifuged at 4000g for 20 minutes in plasma extraction. Epididymal, perirenal, and retroperitoneal fat pads from the abdominal cavity were collected for total abdominal/visceral fat.Data from the first and second studies were analyzed as 2x3 and 2x2 factorials, respectfully, using the MIXED procedure of SAS Version 9.3 . Exceptions include pre-intervention data from the first study used to plan the second study, which were evaluated using student t-tests in Excel. The MIXED procedure was used to assure equal variance, normal distribution, and to identify outliers. Any observations that were more than three standard deviations away from the mean were considered outliers. Four total data points were removed: 28R1 and 28R5 were removed from GLP-1A and fasting BG measurements from the first study; KO11 and WTC9 from the second study’s insulin and leptin measurements, respectively. WTR/S1 and WTR/S2 were removed from food intake data due to suspected equipment malfunction. Data were transformed by log if the normality assumption was not met. This was followed by F-protected least significant difference mean comparison tests in order to determine differences among dietary treatments. Results were considered significant at p<0.05 and expressed as means ± standard error.Sitagliptin was tested in the regulation of adiposity in type 2 diabetes mice, which was generated by a single injection of STZ into diet-induced obese mice. This obese and diabetic model was used in this study to mimic human type 2 diabetes, which is often associated with obesity. Pancreatic islets were partially impaired in the models by STZ to increase blood glucose further in the presence of insulin resistance in DIO mice. It is known that hyperglycemia is not strong in DIO mice. In this mouse model, sitagliptin reduced body weight, body fat content, and visceral fat . The effTo determine the role of GLP-1 in the effect of treatment, GLP-1 receptor knockout (KO) mice were used in this study. The GLP-1R gene was inactivated in KO mice as described elsewhere . The micReduction in adiposity is usually a result of change in energy balance. Energy balance was assessed in mice using metabolic chambers. The treatment increased energy expenditure in mice as suggested by the increased oxygen consumption and carbon dioxide production . The resGLP-1 was examined in blood using the Multiplex kit to understand the mechanism of sitagliptin action. Serum GLP-1 was increased by 50% in WT mice by the treatment . HoweverOur study suggests that administration of sitagliptin through dietary supplementation inhibits adiposity. It was reported in several studies that DPP-4 inhibitors, such as sitagliptin, had no effect on body weight –13. In oThe increased plasma GLP-1 is a potential mechanism of the weight loss. GLP-1 stimulates fat oxidation in both lean and obese conditions . In ratsOur data suggest that GLP-1R may regulate serum level of GLP-1. The circulating GLP-1 concentration reflects a balance of secretion and degradation. The secretion by L-cells is induced by food intake. In this study, sitagliptin induced a greater elevation of GLP-1 in KO mice. GLP-1 secretion is induced by cAMP and ERK1/2 signaling pathways in L-cells, and such as reaction is induced by GLP-1 through activation of the receptor . HoweverFood intake was increased by the treatment of sitagliptin plus resistant starch in this study via an unknown mechanism. One possibility is that the increased energy expenditure from the treatment makes the mice eat more in compensation for the increased calorie demand. Alternatively, the increased may be related to energy dilution by resistant starch in the diet although the diets were prepared to be isocaloric.In summary, we observed that sitagliptin administration through dietary supplementation reduced adiposity and improved glucose metabolism in obese and diabetic mice. Sitagliptin activity in the inhibition of adiposity was enhanced by resistant starch at 28% of diet weight. The combined treatment enhanced energy expenditure in mice, which was associated with GLP-1 elevation. The effects of combined treatment are dependent on GLP-1 as the effects were lost in GLP-R1 KO mice. The activity of sitagliptin was enhanced by resistant starch.